Off-type plants in wheat by aneuploidy
Giura A.1
1
National Agricultural Research and Development Institute Fundulea
*Corresponding author. Email: agiura@ricic.ro
Abstract
Compulsory requirements of varietal purity in wheat tolerate
minimum presence of the atypical variants within a cultivar. If such variants
exceed the admited standards, they cause problems at the seed certification.
We cytologicaly analysed the progeny of atypical tall plants in some semidwarf Romanian cultivars carrying height reducing gene RhtB1 (previous
designation Rht1). The results showed that the tall plants are mostly
aneuploids especially monosomics (2n=41) for chromosome 4B on which the
gene is located.
The increased height of monosomic 4 B plants is the result of reduced dosage
for RhtB1 gene that normally acts in euploids (2n=42) as the height
suppressor. Mitotic analysis on Breeder's stock of recently released doubled
haploid (DH) cultivar Glosa revealed a presence of 0.44% aneuploids and
even of a plantlet with 2n=63.
Before being accepted and officially registered a
new wheat bread cultivar is carefully verified for
agronomic values, distinctness, uniformity, stability
and other traits, in accordance with compulsory
standards and requirements of varietal purity
established at national and EU levels.
In Romania, in conformity with these standards,
only a reduced percentage of atypical plants is accepted
and this varies from 99.9% purity for Prebasic seed to
99.0% at certified C2 stock. To keep the seed stocks as
pure as possible during all steps of field trials and
multiplication chain, also means a certain advantage
for seed trade and promotion of the new variety.
Usually, the off-type, atypical plants occurence is
atributed to accidental contamination with seeds of
other varieties, hazardous outcrossing, spontaneous
mutations, residual heterozygosity and presence of
different biotypes. Even if a great homogeneity can be
now attained by developing doubled haploid (DH)
cultivars, the occurence of deviant phenotypes, as
genetic variants can not be completely avoided. Plants
with different phenotypes could appear as a natural
change in basic chromosome number due to some
specific meiotic disturbances, during micro and
macrosporogenesis (1, 3, 4, 8, 10, 11). Common wheat
(Triticum aestivum L.) is a polyploid species of
complex structure (2n=6x=42) which can tolerates well
both chromosomal deficiency and duplication for each
of the 21 chromosomes.
The aneuploid progenies, i.e. monosomics
(2n=41); nullisomics (2n=40); trisomics (2n=43);
tetrasomics (2n=44), etc., dependent on their frequency
and specific modification of plant morphology can be
Key words
varietal purity, aneuploidy,
height
reducing
gene,
dosage effect
easily confused with other off-types resulted from
outcrosses or mechanical impurifications. Moreover
some types of aneuploids, especially monosomics are
not recognizable as natural genetic variants by the
testing autorities. The aneuploids for chromosome 5A
with speltoid ear (monosomics or nullisomics) and
compactoid ear (trisomics or tetrasomics) are
exceptionally excluded from the evaluation of varietal
impurity in some countries (12).
However, the aneuploid frequency in wheat
cultivars may be higher then officially admited levels
of impurity and this could causes problems at
certification. For example, in five traditional (tall)
wheats, an average 1.08% aneuplolid progeny, ranging
from 1.60% in Hybrid 46 to 0.37% in Cappelle Desprez was found; some values being above the
standards (7).
The introduction of semidwarf varieties at the end
of last century has led to a new reconsideration on the
meaning of "atypical form". The majority of tall plants
which usually occured in these varieties are indeed
aneuploid types - especially monosomics for the
chromosomes 4B and 4D that carry height reducing
gene RhtB1 and respectively RhtD1 (previoulsy Rht2).
The increased height of monosomic plants is the results
of dosage effect of RhtB1/rhtB1 and RhtD1/rhtD1 loci.
Such types of aneuploids can not easily be
distinguished from unacceptable deviants and add new
question on the matter of seed certification.
In Basic seed of the English semi-dwarf variety
Brigand with RhtD1 gene a rate of 1.53 aneuploids per
thousand plants was found, this value exceeding the
total level of off-types permissive in the population
5
(12). Even higher frequencies of aneuploids, from
5.77% to 10.42% were registered in some Argentinian
semi-dwarf varieties carrying RhtB1 gene (9).
Outstanding differences of aneuploid frequencies
between years of storage in the LasRosas-INTA
cultivar were also noted (9).
A preliminary evaluation on aneuploids presence
in three Romanian wheats carrying RhtB1 gene
revealed frequencies ranged from 0.08% to 0.10%
reaching the standard limits of varietal purity (2).
This paper presents new data concerning off-types
frequencies in Romanian wheats and some
morphological characteristics of monosomics that
differentiate them from euploids.
Material and Methods
Plant material is represented by four semi-dwarf
winter wheat varieties Fundulea 133, Fundulea 4,
Dropia and Glosa which were released at the National
Agricultural Research and Development Institute
Fundulea. These varieties carry RhtB1 gene for
reducing height. The translocation 1B/1R is present in
Fundulea 4, uncertain in Fundulea 133 and absent in
Dropia and Glosa. Samples of Breeder's seed and
Prebasic I stocks were grown in different years in field
plots as spaced plants. After anthesis off-type plants
were easily identified by their taller distinctive
phenotype. The plant chromosome complement was
establised by means of chromosome counts on root-tip
squashes stained by Feulgen method. The acetocarmine
anther smear technique was used to check the
monosome 4B identity on the F1 hybrids between
monosomic plants and Chinese Spring ditelocentric
4BL tester line.
An experimental field layout consisting of single
plant randomization design was used to assess the
variation in height, tiller number and other plant traits
of monosomics and disomics (euploids).
Results and Discussions
Field observation in seed multiplication plots of
semi-dwarf Romanian cultivars showed the presence of
atypical plants easily recognizable by their height after
anthesis.
All of the four cultivars produced tall off-types
with various frequencies in different years and
multiplication steps, too. Generally an increased offtype plant number was found in the second and third
year of the experiment, both in Breeder's seed and
Prebasic I plots (Table 1). Such tall plants could be
resulted de novo as aneuploids for chromosome 4B by
abnormal chromosome pairing due to the temperature
variation during meiosis (5; 6). Other factors could also
contribute to the karyotypic instability and taller plants
occurence like precocious ageing of seed due to
improper storage condition (10). The size of the seeds
after seed conditioned process may be another factor,
because a higher percent of aneuploids was found in
wrinkled ones (9) and, not less important factor, the
correctness of purification process in previous seed
multiplication fields.
Table 1
Tall off-type plants frequency in verification plots (different years)
Fundulea 133
Breeder's seed
Prebasic I
Estimated population
off-types
Estimated population
of-types
no.
no.
no.
%
no.
%
7805
4
0.051
11080
6
0.054
6980
7
0.100
7080
8
0.113
3390
4
0.118
Fundulea 4
10905
8
0.073
18095
15
0.083
19765
21
0.106
16605
18
0.108
30620
35
0.114
Dropia
2507
2
0.080
4268
5
0.117
6435
6
0.093
Glosa
3312
3
0.081
7432
8
0.107
-
6
Random samples of tall plants of Fundulea 133
and Fundulea 4 were grouped in two height classes and
seeds of individual plants were then cytologically
analysed. The majority of the analysed plants in taller
classes were monosomics (Table 2). Only three plants
out of 20 were monosomics (2n=41) in the class of 9196 cm while in the class of 97-104 cm, 23 plants out of
29 proved to be monosomics.
A similar high proportion of monosomics (85.0%)
was found among taller plants of Fundulea 4.
Table 2
Euploid and aneuploid frequencies in taller classes of plants
Cultivar
Fundulea 133
Fundulea 4
Classes of
height (cm)
91 - 96
97 - 104
92 - 103
Analysed
plant (no)
20
29
25
105 - 115
20
Meiotic analysis of F1 hybrids (monosomics x
ditelocentric 4BL tester line) validated the 4B
monosomic identity of tall analyzed aneuploids.
The presence of euploid taller plants is difficult to
explain. They could be a result of outcrosses during
seed multiplication or have originated from accidental
contamination. Another explanation is that the
Fundulea 4 may contain a rare biotype with different
photoperiod reaction and vernalization requirements
reflected in an early spring development that could
influence the final plant height (2). A misclassification
was also possible since some plants classified as
Euploids
(no; %)
17 (85.0)
6 (20.7)
18 (72.0)
3 (15.0)
Aneuploids
(no; %)
3 (15.0)
23 (79.3)
7 (28.0)
17 (85.0)
disomics could be monotrisomics or carry other
combinations of 2n=42 chromosomes (9). On the other
hand, a small part of chromosome 4B carrying the
dwarfing RhtB1 gene might be lost.
The cytologically selected monosomics of
Fundulea 133 and Fundulea 4 together with normal
disomic plant were studied in the field using the single
plant randomization design. The results of
morphological measurements revealed significant
differences between euploid and monosomic plants
regarding plant height, yield per plant and some spike
characteristics (Table 3).
Table 3
Some traits of monosomics in comparison with euploids in semi-dwarf winter wheats
Fundulea 133 : 34 disomics; 26 monosomics
Traits
Culm height (cm)
No. of tillers
Mean spike lenght (cm)
Euploids
92.13±1.04
12.12±1.03
9.01±0.11
Monosomics 4B
100.0±0.55
10.61± 0.67
8.88±0.14
No. of spikelets
No. of seeds
Seed weight/mean spike (g)
Yeld per plant (g)
20.09±0.15
42.97±1.21
1.64±0.04
11.8±0.90
19.04±0.22
35.92±1.80
1.38±0.07
8.13±0.66
-1.05***
-7.05**
-1.02***
-3.74***
108.33±1.05
7.08±0.67
9.93±0.17
20.75±0.23
30.54±1.87
1.40±0.11
8.32±1.40
14.48***
0.25ns
0.21ns
-0.89**
-21.71***
-1.02***
-4.16**
Difference
7.87***
1.51ns
0.13ns
Fundulea 4 : 36 disomics; 24 monosomics
Culm height (cm)
No. of tillers
Mean spike lenght (cm)
No. of spikelets
No. of seeds
Seed weight/mean spike (g)
Yeld per plant (g)
ns - not significant
** P<0.01;
*** P<0.001
93.85±1.03
6.83±0.52
10.14±0.010
21.64±0.14
52.25±1.63
2.42±0.09
12.48±1.13
7
The effect of monosomy for chromosome 4B
compared to euploids was the culm height significant
increase with 7.87 cm in Fundulea 133 and
respectively with 14.48 cm in Fundulea 4. This means
that chromosome 4 B in Fundulea 133 has less effect
on height than in Fundulea 4. Different RhtB1 gene
effect on height could also be explained by the
interaction with other background controlling height
genes, or to the presence of different allelic variants at
RhtB1 locus.
The differences between monosomics and
disomics for the mean spike lenght and the number of
spikelets per spike were not significant in both
cultivars. Contrary, the number of grain per spike, seed
weight per spike and yield per plant were all negatively
influenced by monosomic condition being significantly
lower in monosomics.
Although seed producers take careful measures to
eliminate any atypicall plants during seed
multiplication chain, some aneuploids may escape and
perpetuate the specific aneuploid type to the next
generation. In the progeny of selfed monosomic mean
spike we found a monosomic frequency ranging from
56.14% in Fundulea 4 to 61.82% in Dropia. The
nullisomic frequency varied from 3.51% in Fundulea 4
to 6.45% in Fundulea 133 (Table 4).
The presence of recognizable aneuploids for
chromosome 4B in Breeder's seed and Prebasic I plant
population of the analysed cultivars led us to the
presumption that overall aneuploid frequency should
be higher if other chromosomes were lost at a similar
rate. In order to test this hypothesis a mitotic analysis
was carried out on Breeder's seed of Glosa variety. Out
of 451 analysed seeds 448 were euploids, one plant
was monosomic (2n=41) one plant trisomic (2n=43)
and another one had 63 chromosome, probably resulted
by pollination of a non reduced meiocyte with a normal
grain pollen.
Aneuploid frequency of 0.44% that we found in
analysed seeds of Glosa Breeder's stock did not exceed
the admissible stndards of varietal purity at this seed
category. The identity of monosomic and trisomic
plants has not yet been established. However, it is
worth to mention that the field plot was purified after
anthesis and off-types eliminated before harvest time.
Table 4
Euploid and aneuploid progeny of the main spike in 4B monosomics
Cultivar
Fundulea 133
Fundulea 4
Dropia
No. germinated seeds
Euploids
2n=42
Monosomics
2n=41
Nullisomics
2n=40
62
57
55
20 (32.26%)
23 (40.35%)
19 (34.55%)
38 (61.29%)
32 (56.14%)
34 (61.82%)
4 (6.45%)
2 (3.51%)
2 (3.64%)
Conclusions
1.
2.
3.
Aneuploids occurence in common wheat
cultivar population should be considered a
natural genetic variant and treated in an
adequate manner.
Among the tall off-type group of plants found
in Romanian cultivars the majority were
aneuploids for chromosome 4B which carry
RhtB1 height reducing gene.
The seed producers should take all necessary
measures to keep the seed stocks as pure as
possible during multiplication chain to prevent
any troubles at the seed certification.
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